Enzyme with protease activity

ABSTRACT

A DNA construct comprising a DNA sequence encoding an enzyme exhibiting protease activity, which DNA sequence comprises the DNA sequence shown in SEQ ID No. 1 or 2 or an analog of any of these sequences being at least 80% homologous to the DNA sequence shown in SEQ ID No. 1 or 2. The proteases encoded by the DNA sequences have an acid pH optimum.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of PCT/DK94/00274 filed Jul. 5, 1994,which is incorporated herein by reference.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of PCT/DK94/00274 filed Jul. 5, 1994,which is incorporated herein by reference.

FIELD OF INVENTION

The present invention relates to a DNA construct encoding an enzyme withprotease activity, a method of producing the enzyme, an enzyme withprotease activity, and an enzyme preparation containing the enzyme.

BACKGROUND OF THE INVENTION

Proteases are enzymes capable of cleaving peptide bonds. Acid proteases(i.e. proteases having an acidic pH optimum) have been found to beproduced by a number of different organism including mammals andmicrobes. For instance, microbial acid proteases have been found to beproduced by bacterial strains such as strains of Bacillus sp. (JP01240184), fungal strains, e.g. of Rhizopus sp. (EP 72978), Schytalidiumsp. (JP 48091273), Sulpholobus sp. and Thermoplasma sp. (WO/90 10072)and Aspergillus sp. (JP 50121486, EP 82 395).

JP 3058794 discloses the cloning of a gene encoding an acid proteasefrom R. niveus and the recombinant expression thereof. The cloning andexpression of a gene from Cryphonectira parasitica encoding an asparticprotease is described by Choi et al. (1993). Takahashi et al. (1991),Inoue et al. (1991), and JP 407 5586 discloses the cloning of a genefrom Aspergillus niger encoding an acid proteinase (Protease A).

Berka et al. (1990) disclose a gene encoding the aspartic proteinaseaspergillopepsin A from Aspergillus awamori. The cloning of a geneencoding the aspartic proteinase aspergillopepsin O from Aspergillusoryzae is described by Berka et al. (1993). The cloning of a geneencoding the acid protease (PEPA) from Aspergillus oryzae is disclosedby Gomi et al. (1993).

Acid proteases are widely used industrially, e.g. in the preparation offood and feed, in the leather industry (e.g. to dehair hides), in theproduction of protein hydrolysates and in the wine making and brewingindustry.

There is a need for single-component acid proteases for many differentapplications, especially in the food and feed industry.

SUMMARY OF THE INVENTION

It is an object of the present invention to prepare a single-componentprotease.

Accordingly, in a first aspect the invention relates to a DNA constructcomprising a DNA sequence encoding an enzyme exhibiting proteaseactivity, which DNA sequence comprises the DNA sequence shown in SEQ IDNo. 1 or an analogous sequence thereof being at least 80% homologous tothe DNA sequence shown in SEQ ID No. 1.

In a second aspect the invention relates to a DNA construct comprising aDNA sequence encoding an enzyme exhibiting protease activity, which DNAsequence comprises the DNA sequence shown in SEQ ID No. 2 or ananalogous sequence thereof being at least 80% homologous to the DNAsequence shown in SEQ ID No. 2.

The DNA sequence shown in SEQ ID No. 1 encodes an enzyme which in thefollowing disclosure is referred to as Protease I. The enzyme encoded bythe DNA sequence shown in SEQ ID No. 2 is referred to as Protease II.

By a database homology search it has been found that the DNA sequenceshown in SEQ ID Nos. 1 and 2 are generally novel. The highest homologyof the DNA sequence shown in SEQ ID No. 1 to known protease genes wasfound to be 74.7% to the Aspergillus niger acidic proteinase A asdetermined for an overlap of 538 nucleotides. The highest homology toprotease II was found to be 75.5% to the Aspergillus oryzaeaspergillopepsin O as determined for an overlap of 343 nucleotides.

In further aspects the invention relates to an expression vectorharbouring a DNA construct of the invention, a cell comprising the DNAconstruct or expression vector and a method of producing an enzymeexhibiting protease activity which method comprises culturing said cellunder conditions permitting the production of the enzyme, and recoveringthe enzyme from the culture.

In a still further aspect the invention relates to an enzyme exhibitingprotease activity, which enzyme is encoded by a DNA construct of theinvention as defined above or produced by the method of the invention.

In a further important aspect the invention relates to an enzyme withprotease activity, which is active at a pH below 7.0 and in the presenceof up to 3% hydrogen peroxide. In the present context, the term "isactive" as used about the enzyme is intended to indicate that the enzymeis capable of hydrolysing a substrate under the above-mentionedconditions, e.g. as described in example 5 herein.

This enzyme of the invention which is active at a pH below 7.0 and inthe presence of up to 3% hydrogen peroxidase and which removes more than80% of the lysozyme from the lenses under the conditions specified inexample 5 is termed the "H₂ O₂ -stable protease" in the followingdisclosure. This enzyme is believed to be generally novel.

Also, the present invention provides an enzyme with protease activity,which enzyme is active at a pH below 7.0 and which is specific towardsPhe-Val or Lys-Tyr linkages. The term "specific" is intended to indicatethat the enzyme, when the substrate is bovine glucagon, primarilycleaves these linkages.

Protease I and protease II described herein are preferred examples of anenzyme of the invention. The enzymes have been found to be acidproteases, i.e. proteases which has an acid pH optimum.

By the present invention it is possible to provide the protease in ahighly purified form, i.e. greater than 75% pure, and more preferablygreater than 90% pure as determined by SDS gel electrophoresis asdescribed in the Materials and Methods section herein.

In final aspects the invention relates to an enzyme preparationcomprising an enzyme of the invention and the use of the enzyme orenzyme preparation for various purposes in which modification ordegradation of protein-containing substances is desirable.

DETAILED DESCRIPTION OF THE INVENTION

The DNA construct, vector and method of the invention

In the present context the term "analogue" used to define the DNAconstruct of the invention is understood to include any DNA sequencewhich encodes an enzyme with protease activity and which is at least 80%homologous to the DNA sequence shown in SEQ ID No. 1 or 2, respectively.The analogous DNA sequence may be a DNA sequence which hybridizes to thesame probe as the DNA coding for the protease enzyme under the followingconditions: presoaking in 5×SSC and prehybridizing for 1 h at ˜55° C. ina solution of 533 SSC, 5×Denhardt's solution, 50 mM sodium phosphate, pH6.8, and 50 μg of denatured sonicated calf thymus DNA, followed byhybridization in the same solution supplemented with 50 μCi 32-P-dCTPlabelled probe for 18 h at ˜55° C. followed by washing three times in2×SSC, 0.2% SDS at 55° C. for 30 minutes. The analogous DNA sequence ispreferably at least 90% homologous to the sequence shown in SEQ ID No. 1or 2, preferably at least 95% homologous to said sequence.

The analogous DNA sequence may, e.g., be isolated from another organismor may be one prepared on the basis of the DNA sequence shown in SEQ IDNo. 1 or 2, such as by introduction of nucleotide substitutions which donot give rise to another amino acid sequence of the protease but whichcorrespond to the codon usage of the host organism into which the DNAconstruct is introduced or nucleotide substitutions which do give riseto a different amino acid sequence and therefore, possibly, a differentprotein structure which might give rise to a protease mutant withdifferent properties than the native enzyme. Other examples of possiblemodifications are insertion of one or more nucleotides into thesequence, addition of one or more nucleotides at either end of thesequence, or deletion of one or more nucleotides at either end or withinthe sequence.

Furthermore, it is preferred that the protease encoded by the analogousDNA sequence is immunologically cross-reactive with an antibody raisedagainst a purified protease encoded by the DNA sequence shown in SEQ IDNo. 1 or 2.

The nucleotide probe with which the analogue of the DNA sequence shownin SEQ ID No. 1 can hybridize may, e.g. be prepared on the basis of anyof the following DNA sequences or any combination thereof:

(a) AATTAAGCAT CCTCCATCTT (SEQ ID NO: 3)

(b) CAAAGCTCAA TCTCGCTAAC (SEQ ID NO: 4)

(c) TCCCGCTCTT CTCTCGATCT (SEQ ID NO: 5)

(d) CATCATCCCA ATAACTCGGA (SEQ ID NO: 6)

(e) CAAAATGAAG ACCTCTGCTC (SEQ ID NO: 7)

(f) TCTTGACCGC TGGCCTGTTG (SEQ ID NO: 8)

(g) GCACCGCTGC TATTGCTGCT (SEQ ID NO: 9)

(h) CCTCTCACCG CGAAGCGCGC (SEQ ID NO: 10)

(i) ACGTGCTCGC GCTGCCAAGC (SEQ ID NO: 11)

(j) TGGCACCAGC CGCAAGAGCA (SEQ ID NO: 12)

(k) AGGGGGGTCT CAAGCCCGGC (SEQ ID NO: 13)

(l) ACCCAGCGAG GCCATAACCT (SEQ ID NO: 14)

(m) GACCGGCTCC AAGAACACCG (SEQ ID NO: 15)

(n) GAGGTACTCG TCCAACTGGG (SEQ ID NO: 16)

(o) CCGGCGCCGT GCCAT (SEQ ID NO: 17) ##STR1##

These sequences constitute partial sequences of the DNA sequence shownin SEQ ID No. 1 or analogues of such sequences.

The nucleotide probe with which the analogue of the DNA sequence shownin SEQ ID No. 2 can hybridize may, e.g., be prepared on the basis of anyof the following DNA sequences or any combination thereof:

(p1) CTGCTTCTCC TTCTCTTCCT (SEQ ID NO: 19)

(q) CCTCGTGATA TCTGCTTGAA (SEQ ID NO: 20)

(r) CATCTCCTCA TCATGGTCGT (SEQ ID NO: 21)

(s) CCTCAACAAG GTGCAGCCTT (SEQ ID NO: 22)

(t) CTTCTGGGTC TGACCACCGC (SEQ ID NO: 23)

(u) CGCCACTGGT CCCCTGGCCG (SEQ ID NO: 24)

(v) AGCCGCAGGC TTCTGTCCGG (SEQ ID NO: 25)

(w) TCAAGAACTT CTCCGTCAAG (SEQ ID NO: 26)

(x) CAGGTCGAGA AGGCGGGCAG (SEQ ID NO: 27)

(y) CAAGGGACGT ACCGTTAACC (SEQ ID NO: 28)

(z) TGCCGGGTCT GTATGCGAAT (SEQ ID NO: 29)

(aa) GCGCTGGCCA AGTATGGCGC )SEQ ID NO: 30)

(bb) CCAGGTGCGG CCAGCGTCAA (SEQ ID NO: 31)

(cc) GGCCGCCGCC GTCAGTGGCA (SEQ ID NO: 32)

(dd) GCGTCGTGAC CACCCGCAGG CCAACGACG (SEQ ID NO: 33) ##STR2##

These sequences constitute partial sequences of the DNA sequence shownin SEQ ID No. 2 or analogues of such sequences.

A DNA sequence of the invention may be isolated by a general methodinvolving

cloning, in suitable vectors, a DNA library from Aspergillus aculeatus,

transforming suitable yeast host cells with said vectors,

culturing the host cells under suitable conditions to express any enzymeof interest encoded by a clone in the DNA library, and

screening for positive clones by determining any protease activity ofthe enzyme produced by such clones.

A more detailed description of this screening method is given in Example1 below and in WO 93/11249, the contents of which is hereby incorporatedby reference.

The DNA sequence coding for the enzyme may for instance be isolated byscreening a cDNA library of Aspergillus aculeatus, e.g strain CBS101.43, publicly available from the Centraalbureau voorSchimmelcultures, Delft, NL, and selecting for clones expressing theappropriate enzyme activity (i.e. protease activity as defined by theability of the enzyme to hydrolyse peptide bonds in proteins andpeptides). The appropriate DNA sequence may then be isolated from theclone by standard procedures, e.g. as described in Example 1.

It is expected that a DNA sequence coding for a homologous enzyme, i.e.an analogous DNA sequence, may be derived by similarly screening a cDNAlibrary of another microorganism, in particular a fungus, such as astrain of another Aspergillus sp., in particular a strain of A.aculeatus or A. niger, a strain of a Trichoderma sp., in particular astrain of T. harzianum, or T. reesie, a strain of a Fusarium sp., inparticular a strain of F. oxysporum, a strain of Rhizopus sp., e.g. R.niveus, a strain of Schytalidium sp., or a strain of a Humicola sp.

Alternatively, the DNA sequence of the invention may, in accordance withwell-known procedures, conveniently be isolated from DNA from anappropriate organism by use of synthetic oligonucleotide probes preparedon the basis of a DNA sequence disclosed herein. For instance, asuitable oligonucleotide probe may be prepared on the basis of any ofthe partial nucleotide sequences shown above.

The DNA sequence may subsequently be inserted into a recombinantexpression vector. This may be any vector which may conveniently besubjected to recombinant DNA procedures, and the choice of vector willoften depend on the host cell into which it is to be introduced. Thus,the vector may be an autonomously replicating vector, i.e. a vectorwhich exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g. a plasmid. Alternatively,the vector may be one which, when introduced into a host cell, isintegrated into the host cell genome and replicated together with thechromosome(s) into which it has been integrated.

In the vector, the DNA sequence encoding the protease should be operablyconnected to a suitable promoter and terminator sequence. The promotermay be any DNA sequence which shows transcriptional activity in the hostcell of choice and may be derived from genes encoding proteins eitherhomologous or heterologous to the host cell. The procedures used toligate the DNA sequences coding for the protease, the promoter and theterminator, respectively, and to insert them into suitable vectors arewell known to persons skilled in the art (cf., for instance, Sambrook etal., Molecular Cloning. A Laboratory Manual, Cold Spring Harbor, N.Y.,1989).

The host cell which is transformed with the DNA sequence encoding theenzyme of the invention is preferably a eukaryotic cell, in particular afungal cell such as a yeast or filamentous fungal cell. In particular,the cell may belong to a species of Aspergillus, most preferablyAspergillus oryzae or Aspergillus niger. Fungal cells may be transformedby a process involving protoplast formation and transformation of theprotoplasts followed by regeneration of the cell wall in a manner knownper se. The use of Aspergillus oryzae as a host microorganism isdescribed in EP 238 023 (of Novo Noridisk A/S), the contents of whichare hereby incorporated by reference. The host cell may also be a yeastcell, e.g. a strain of Saccharomyces, in particular Saccharomycescerevisiae, Saccharomyces kluyveri or Saccharomyces uvarum, a strain ofSchizosaccaromyces sp., such as Schizosaccharomyces pombe, a strain ofHansenula sp., Pichia sp., Yarrowia sp. such as Yarrowia lipolytica, orKluyveromyces sp. such as Kluyveromyces lactis.

In a still further aspect, the present invention relates to a method ofproducing an enzyme with protease activity, wherein a suitable host celltransformed with a DNA construct of the invention encoding the enzyme iscultured under conditions permitting the production of the enzyme, andthe resulting enzyme is recovered from the culture.

The medium used to culture the transformed host cells may be anyconventional medium suitable for growing the host cells in question. Theexpressed protease may conveniently be secreted into the culture mediumand may be recovered therefrom by well-known procedures includingseparating the cells from the medium by centrifugation or filtration,precipitating proteinaceous components of the medium by means of a saltsuch as ammonium sulphate, followed by chromatographic procedures suchas ion exchange chromatography, affinity chromatography, or the like.

The enzyme of the invention

Preferably, the protease of the invention (e.g. the H₂ O₂ -stable or theLys-Tyr or Phe-Val specific protease) is active at a pH in the range of2-7, such as at a pH below 6.0, e.g. in the range of 2-6, and mostpreferably in the pH range of 4-6.

It will be understood that both acidic proteases are active in thepresence of 0.01-5% hydrogen peroxide, such as 0.1-5%, 0.5-4%, 1-4% or2-3% hydrogen peroxide and are applicable to contact lens cleaning.

A preferred example of an H₂ O₂ -stable protease of the invention is theenzyme encoded by the DNA sequence shown in SEQ ID No. 1 or an analoguethereof as defined above, which is at least 80% homologous to said DNAsequence.

A preferred example of the Lys-Tyr or Phe-Val specific protease of theinvention is the enzyme encoded by the DNA sequence shown in SEQ ID No.2 or an analogue thereof as defined above, which is at least 80%homologous to said DNA sequence.

The enzyme of the invention is preferably immunologically reactive withan antibody raised against a purified protease derived from Aspergillusaculeatus, CBS 101.43 and being encoding by the DNA sequence shown inSEQ ID No. 1 or 2. In the present context, the term "derived from" isintended not only to indicate a protease produced by strain CBS 101.43,but also a protease encoded by a DNA sequence isolated from strain CBS101.43 and produced in a host organism transformed with said DNAsequence.

While the H₂ O₂ -stable protease and the Lys-Tyr or Phe-Val specificprotease of the invention both were obtained from a strain of the fungalspecies Aspergillus aculeatus, it is contemplated that such enzymes areobtainable from other organisms as well, in particularly microorganisms.

Thus, the enzyme of the invention is preferably obtainable from abacterium or a fungus such as a strain of Aspergillus, Rhizopus,Trichoderma,, e.g. T. reesei or T. harzianum, Penicillium, Fusarium,Schytalidium or Humicola, e.g. H. insolens or H. lanuginosa, or a strainof Bacillus.

Examples of Aspergillus sp. include A. niger, A. oryzae or A. aculeatus,such as A. aculeatus CBS 101.43.

In a still further aspect, the present invention relates to an enzymepreparation useful for the degradation or modification of proteasecontaining materials, said preparation being enriched in an enzymeexhibiting protease activity as described above.

The enzyme preparation having been enriched with an enzyme of theinvention may e.g. be an enzyme preparation comprising multipleenzymatic activities, in particular an enzyme preparation comprisingmultiple plant cell wall degrading enzymes such as Pectinex®, PectinexUltra SP®, Gamanase, Celluclast or Celluzyme or protease and/orexopeptidase-containing enzyme preparations such as Neutrase®, Alcalase®or Flavourzyme® (all available from Novo Nordisk A/S). In the presentcontext, the term "enriched" is intended to indicate that the proteaseactivity of the enzyme preparation has been increased, e.g. with anenrichment factor of at least 1.1, conveniently due to addition of anenzyme of the invention prepared by the method described above.

Alternatively, the enzyme preparation enriched in an enzyme exhibitingprotease activity may be one which comprises an enzyme of the inventionas the major enzymatic component, e.g. a mono-component enzymepreparation.

The enzyme preparation may be prepared in accordance with methods knownin the art and may be in the form of a liquid or a dry preparation. Forinstance, the enzyme preparation may be in the form of a granulate or amicrogranulate. The enzyme to be included in the preparation may bestabilized in accordance with methods known in the art.

The enzyme preparation according to the invention may be used as anagent for degradation or modification of plant cell walls. Someproteins, like extensins, are components of plant cell walls. Proteaseswill therefore facilitate the degradation or modification of plant cellwalls. Such a protease containing plant cell wall degrading enzymepreparation can be used for many different applications like extractionof oil from plant sources like olives and rape or for production ofjuice from different fruits like apples, pears and citrus.

The enzyme preparation may additionally contain one or more other plantcell wall degrading enzymes such as a pectin lyase, pectate lyase,endoglucanase, arabinanase, xylanase, glucanase, galactanase, mannanase,α-galactosidase, rhamnogalacturonase, pectin acetylesterase,polygalacturonase, protease, exo-peptidase or pectin methylesterase. Thepreparation may further contain one or more enzymes exhibitingexo-activity on the same substrates as the above-mentioned endo-enzymes.The proteases according to the invention work at the same pH andtemperature conditions as many other cell wall degrading enzymes, andare thereby particular well suited for such applications.

The additional enzyme(s) may be producible by means of a microorganismbelonging to the genus Aspergillus, preferably Aspergillus niger,Aspergillus aculeatus, Aspergillus awamori or Aspergillus oryzae, orTrichoderma.

The protease enzyme preparation according to the invention may also beused in the wine industry, to prevent haze or to dissolve haze, asproteins often take part in the undesirable haze formation. Theproteases according to the invention are active under the conditionspresent under fermentation and maturation of wine, and they aretherefore particular useful for this application.

The enzyme or enzyme preparation of the invention may be used in baking,e.g. in order to weaken the gluten components of flour so as to obtain asoftening of so-called hard flour. The use of weak flour is importantfor the preparation of dough which must be very extensible and notelastic, e.g. in the preparation of extruded baked products, biscuitsand other products which must keep their original shape during transportand baking. The proteases of the invention constitute a desirablealternative to conventionally used agents for weakening of flour, suchas sodium metabisulphite (SMS). The use of SMS is considered to beundesirable because of potential health risks.

The protease preparation may also be used in the food and feed industryto improve the digestibility of proteins. For instance, the enzyme orenzyme preparation may be added to animal feed or may be used to processanimal feed, in particular feed for piglets or poultry. Thereby, thedigestibility of components of the feed may be increased resulting in animproved growth rate and efficiency of feed utilisation of the animals,cf. Brenes et al. (1993).

Further the enzyme or enzyme preparation of the invention may be usefulto make protein hydrolysates from, e.g., vegetable proteins like soy,pea, lupin or rape seed protein, milk like casein, meat proteins, orfish proteins. The protease may be used for protein hydrolysates toimprove the solubility, consistency, taste, or fermentability, to reduceantigenicity or for other purposes to make food, feed or dedicalproducts. The protease may be used alone or together with otherproteases or together with other enzymes like exopeptidases. The use ofthe protease of the invention together with exopeptidase rich enzymepreparations will improve the taste of the protein hydrolysates.

The protease preparation may also be used to modify proteins, likereducing viscosity caused or partially caused by proteins. Suchviscosity problems are known in the processing of different proteincontaining plant materials like soya and peas.

Furthermore, the enzyme or enzyme preparation may be used in theprocessing of fish or meat, e.g. to change texture and/or viscosity.

The protease preparation may also be used to facilitate fermentativeprocesses, like yeast fermentation of barley, malt and other rawmaterials for the production of e.g. beer.

Furthermore, the enzyme or enzyme preparation of the invention may beuseful in the leather industry e.g. to remove hairs from hides. The lowpH optimum of the protease is an advantage as the subsequent tanning ofthe hides is carried under acid conditions. The protease preparation isuseful for production of peptides from proteins, where it is advantagesto use a cloned enzyme essentially free from other proteolyticactivities.

Further the protease preparation can be used to degrade protein in orderto facilitate purification of or to upgrade different products, like inpurification or upgrading of gums, like guar gum, xanthan gum, degummingof silk, or improvement of the quality of wool.

Due to the stability towards hydrogen peroxide both proteases of theinvention are of particular use for cleaning of contact lenses and otherapplications involving the use of hydrogen peroxide, in whichprotein-containing material is to be removed.

For the above uses, the dosage of the enzyme preparation of theinvention and other conditions under which the preparation is used maybe determined on the basis of methods known in the art.

The invention is further described in the accompanying drawing in which

FIG. 1 illustrates the pH activity profiles of Protease I and II,respectively,

FIG. 2 the temperature-activity profiles of Protease I and II,respectively,

FIGS. 3 and 4 the pH stability of Protease I and II, respectively,

FIG. 5 and 6 the temperature stability of Protease I and II,respectively,

FIG. 7 the performance of various proteases in the cleaning of contactlenses, and

FIG. 8 the gluten stretching effect of an enzyme of the invention.

The invention is described in further detail in the following exampleswhich are not in any way intended to limit the scope of the invention asclaimed.

MATERIALS AND METHODS

Donor organism: mRNA was isolated from Aspergillus aculeatus, CBS101.43, grown in a soy-containing fermentation medium with agitation toensure sufficient aeration. Mycelia were harvested after 3-5 days'growth, immediately frozen in liquid nitrogen and stored at -80° C.

Yeast strains: The Saccharomyces cerevisiae strain used was yNG231 (MATalpha, leu2, ura3-52, his4-539, pep4-delta 1, cir+) or JG169 (MATα; ura3-52; leu 2-3, 112; his 3-D200; pep 4-113; prc1::HIS3; prb1:: LEU2;cir+).

Plasmids: The expression plasmid pYHD17 containing the yeast TPIpromoter was prepared from the commercially available plasmid PYES II(Invitrogen). The plasmid and the construction thereof is furtherdescribed in WO 93/11249, the contents of which is hereby incorporatedby reference.

The Aspergillus expression vector pHD414 is a derivative of the plasmidp775 (described in EP 238 023). The construction of pHD414 is furtherdescribed in WO 93/11249. pHD414 contains the A. niger glucoamylaseterminator and the A. oryzae TAKA amylase promoter.

Extraction of total RNA: The total RNA was prepared by extraction withguanidinium thiocyanate followed by ultracentrifugation through a 5.7MCsCl cushion essentially as described by Chirgwin et al., 1979 and in WO93/11249.

Isolation of poly(A)⁺ RNA: The poly(A)⁺ RNAs were isolated byoligo(dT)-cellulose affinity chromatography (Aviv & Leder, 1972).Typically, 0.2 g of oligo(dT) cellulose (Boehringer Mannheim) waspreswollen in 10 ml of 1×column loading buffer (20 mM Tris-Cl, pH 7.6,0.5M NaCl, 1 mM EDTA, 0.1% SDS), loaded onto a DEPC-treated, pluggedplastic column (Poly Prep Chromatography Column, Bio Rad), andequilibrated with 20 ml 1×loading buffer. The total RNA was heated at65° C. for 8 min., quenched on ice for 5 min, and after addition of 1vol 2×column loading buffer to the RNA sample loaded onto the column.The eluate was collected and reloaded 2-3 times by heating the sample asabove and quenching on ice prior to each loading. The oligo(dT) columnwas washed with 10 vols of 1×loading buffer, then with 3 vols of mediumsalt buffer (20 mM Tris-Cl, pH 7.6, 0.1M NaCl, 1 mM EDTA, 0.1% SDS),followed by elution of the poly(A)⁺ RNA with 3 vols of elution buffer(10 mM Tris-Cl, pH 7.6, 1 mM EDTA, 0.05% SDS) preheated to +65° C., bycollecting 500 μl fractions. The OD₂₆₀ was read for each collectedfraction, and the mRNA containing fractions were pooled and ethanolprecipitated at -20° C. for 12 h. The poly(A)⁺ RNA was collected bycentrifugation, resuspended in DEPC-DIW and stored in 5-10 μg aliquotsat -80° C.

Northern blot analysis: The poly(A)⁺ RNAs (5 μg/sample) from variousmycelia were electrophoresed in 1.2 agarose-2.2M formaldehyde gels(Sambrook et al., 1989) and blotted to nylon membranes (Hybond-N,Amersham) with 10×SSC (Sambrook et al., 1989) as transfer buffer. Threerandom-primed (Feinberg & Vogelstein, 1983) ³² P-labeled cDNA probeswere used in individual hybridizations: 1) a 1.3 kb Not I-Spe I fragmentfor polygalacturonase I from A. aculeatus, 2) a 1.3 kb Not I-Spe Ifragment encoding endoglucanase I from A. aculeatus and 3) a 1.2 kb EagI fragment coding for galactanase I from A. aculeatus. Northernhybridizations were carried out in 5×SSC (Sambrook et al., 1989),5×Denhardt's solution (Sambrook et al., 1989), 0.5% SDS (w/v) and 100μg/ml denatured salmon sperm DNA with a probe concentration of ca. 2ng/ml for 16 h at 65° C. followed by washes in 5×SSC at 65° C. (2×15min), 2×SSC, 0.5% SDS (1×30 min), 0.2×SSC, 0.5% SDS (1×30 min), and5×SSC (2×15 min). After autoradiography at -80° C. for 12 h, the probe #1 was removed from the filter according to the manufacturer'sinstructions and rehybridized with probe #2, and eventually with probe#3. The RNA ladder from Bethesda Research Laboratories was used as asize marker.

cDNA synthesis:

First strand synthesis: Double-stranded cDNA was synthesized from 5 μgof A. aculeatus poly(A)⁺ RNA by the RNase H method (Gubler & Hoffman1983, Sambrook et al., 1989) using the hair-pin modification. Thepoly(A)⁺ RNA (5 μg in 5 μl of DEPC-treated water) was heated at 70° C.for 8 min., quenched on ice, and combined in a final volume of 50 μlwith reverse transcriptase buffer (50 mM Tris-Cl, pH 8.3, 75 mM KCl, 3mM MgCl2, 10 mM DTT, Bethesda Research Laboratories) containing 1 mMeach dNTP (Pharmacia), 40 units of human placental ribonucleaseinhibitor (RNasin, Promega), 10 μg of oligo(dT)₁₂₋₁₈ primer (Pharmacia)and 1000 units of SuperScript II RNase H- reverse transcriptase(Bethesda Research Laboratories). First-strand cDNA was synthesized byincubating the reaction mixture at 45° C. for 1 h.

Second strand synthesis: After synthesis 30 μl of 10 mM Tris-Cl, pH 7.5,1 mM EDTA was added, and the mRNA:cDNA hybrids were ethanol precipitatedfor 12 h at -20° C. by addition of 40 μg glycogen carrier (BoehringerMannheim) 0.2 vols 10M NH₄ Ac and 2.5 vols 96% EtOH. The hybrids wererecovered by centrifugation, washed in 70% EtOH, air dried andresuspended in 250 μl of second strand buffer (20 mM Tris-Cl, pH 7.4, 90mM KCl, 4.6 mM MgCl2, 10 mM (NH₄)₂ SO₄, 16 μM BNAD⁺) containing 100 μMeach dNTP, 44 units of E. coli DNA polymerase I (Amersham), 6.25 unitsof RNase H (Bethesda Research Laboratories) and 10.5 units of E. coliDNA ligase (New England Biolabs). Second strand cDNA synthesis wasperformed by incubating the reaction tube at 16° C. for 3 h, and thereaction was stopped by addition of EDTA to 20 mM final concentrationfollowed by phenol extraction.

Mung bean nuclease treatment: The double-stranded (ds) cDNA was ethanolprecipitated at -20° C. for 12 h by addition of 2 vols of 96% EtOH, 0.1vol 3M NaAc, pH 5.2, recovered by centrifugation, washed in 70% EtOH,dried (SpeedVac), and resuspended in 30 μl of Mung bean nuclease buffer(30 mM NaAc, pH 4.6, 300 mM NaCl, 1 mM ZnSO4, 0.35 mM DTT, 2% glycerol)containing 36 units of Mung bean nuclease (Bethesda ResearchLaboratories). The single-stranded hair-pin DNA was clipped byincubating the reaction at 30° C. for 30 min, followed by addition of 70μl 10 nM Tris-Cl, pH 7.5, 1 mM EDTA, phenol extraction, and ethanolprecipitation with 2 vols of 96% EtOH and 0.1 vol 3M NaAc, pH 5.2 at-20° C. for 12 h.

Blunt-ending with T4 DNA polymerase: The ds cDNA was blunt-ended with T4DNA polymerase in 50 μl of T4 DNA polymerase buffer (20 mM Tris-acetate,pH 7.9, 10 mM MgAc, 50 mM KAc, 1 mM DTT) containing 0.5 mM each dNTP and7.5 units of T4 DNA polymerase (Invitrogen) by incubating the reactionmixture at +37° C. for 15 min. The reaction was stopped by addition ofEDTA to 20 mM final concentration, followed by phenol extraction andethanol precipitation.

Adaptor ligation and size selection: After the fill-in reaction the cDNAwas ligated to non-palindromic BstX I adaptors (1 μg/μl, Invitrogen) in30 μl of ligation buffer (50 mM Tris-Cl, pH 7.8, 10 mM MgCl2, 10 mM DTT,1 mM ATP, 25 μg/ml bovine serum albumin) containing 600 pmol BstX Iadaptors and 5 units of T4 ligase (Invitrogen) by incubating thereaction mix at +16° C. for 12 h. The reaction was stopped by heating at+70° C. for 5 min, and the adapted cDNA was size-fractionated by agarosegel electrophoresis (0.8% HSB-agarose, FMC) to separate unligatedadaptors and small cDNAs. The cDNA was size-selected with a cut-off at0.7 kb, and the cDNA was electroeluted from the agarose gel in 10 mMTris-Cl, pH 7.5, 1 mM EDTA for 1 h at 100 volts, phenol extracted andethanol precipitated at -20° C. for 12 h as above.

Construction of cDNA libraries: The adapted, ds cDNA was recovered bycentrifugation, washed in 70% EtOH and resuspended in 25 ml DIW. Priorto large-scale library ligation, four test ligations were carried out in10 μl of ligation buffer (same as above) each containing 1 μl ds cDNA(reaction tubes #1-#3), 2 units of T4 ligase (Invitrogen) and 50 ng(tube #1), 100 ng (tube #2) and 200 ng (tubes #3 and #4) Bst XI cleavedyeast expression vector either pYES 2.0 vector Invitrogen or yHD17). Theligation reactions were performed by incubation at +16° C. for 12 h,heated at 70° C. for 5 min, and 1 μl of each ligation electroporated(200 Ω, 2.5 kV, 25 μF) to 40 μl competent E. coli 1061 cells (OD600=0.9in 1 liter LB-broth, washed twice in cold DIW, once in 20 ml of 10%glycerol, resuspended in 2 ml 10% glycerol). After addition of 1 ml SOCto each transformation mix, the cells were grown at +37° C. for 1 h , 50μl plated on LB+ampicillin plates (100 μg/ml) and grown at +37° C. for12 h.

Using the optimal conditions a large-scale ligation was set up in 40 μlof ligation buffer containing 9 units of T4 ligase, and the reaction wasincubated at +16° C. for 12 h. The ligation reaction was stopped byheating at 70° C. for 5 min, ethanol precipitated at -20° C. for 12 h,recovered by centrifugation and resuspended in 10 μl DIW. One μlaliquots were transformed into electrocompetent E. coli 1061 cells usingthe same electroporation conditions as above, and the transformed cellswere titered and the library plated on LB+ampicillin plates with5000-7000 c.f.u./plate. To each plate was added 3 ml of medium. Thebacteria were scraped off, 1 ml glycerol was added and stored at -80° C.as pools. The remaining 2 ml were used for DNA isolation. If the amountof DNA was insufficient to give the required number of yeasttransformants, large scale DNA was prepared from 500 ml medium (TB)inoculated with 50 μl of -80° C. bacterial stock propagated overnight.

Construction of yeast libraries: To ensure that all the bacterial cloneswere tested in yeast, a number of yeast transformants 5 times largerthan the number of bacterial clones in the original pools was set as thelimit.

One μl aliquots of purified plasmid DNA (100 ng/μl) from individualpools were electroporated (200 Ω, 1.5 kV, 25 μF) into 40 μl competent S.cerevisiae JG 169 cells (OD600=1.5 in 500 ml YPD, washed twice in coldDIW, once in cold 1M sorbitol, resuspended in 0.5 ml 1M sorbitol, Becker& Guarante, 1991). After addition of 1 ml 1M cold sorbitol, 80 μlaliquots were plated on SC+glucose-uracil to give 250-400 c.f.u./plateand incubated at 30° C. for 3-5 days.

Isolation of a cDNA gene for expression in Aspergillus: One or more ofprotease-producing colonies were inoculated into 20 ml YNB-1 broth in a50 ml glass test tube. The tube was shaken for 2 days at 30° C. Thecells were harvested by centrifugation for 10 min. at 3000 rpm.

The cells were resuspended in 1 ml 0.9M sorbitol, 0.1M EDTA, pH 7.5. Thepellet was transferred to an Eppendorf tube, and spun for 30 seconds atfull speed. The cells were resuspended in 0.4 ml 0.9M sorbitol, 0.1MEDTA, 14 mM β-mercaptoethanol. 100 μl 2 mg/ml Zymolase was added, andthe suspension was incubated at 37° C. for 30 minutes and spun for 30seconds. The pellet (spheroplasts) was resuspended in 0.4 ml TE. 90 μlof (1.5 ml 0.5M EDTA pH 8.0, 0.6 ml 2M Tris-Cl pH 8.0, 0.6 ml 10% SDS)was added, and the suspension was incubated at 65° C. for 30 minutes. 80μl 5M KOAc was added, and the suspension was incubated on ice for atleast 60 minutes and spun for 15 minutes at full speed. The supernatantwas transferred to a fresh tube which was filled with EtOH (room temp.)followed by thorough but gentle mixing and spinning for 30 seconds. Thepellet was washed with cold 70% ETOH, spun for 30 seconds and dried atroom temperature. The pellet was resuspended in 50 μl TE and spun for 15minutes. The supernatant was transferred to a fresh tube. 2.5 μl 10mg/ml RNase was added, followed by incubation at 37° C. for 30 minutesand addition of 500 μl isopropanol with gentle mixing. The mixture wasspun for 30 seconds, and the supernatant was removed. The pellet wasrinsed with cold 96% EtOH and dried at room temperature. The DNA wasdissolved in 50 μl water to a final concentration of approximately 100μl/ml.

Transformation of Aspergillus oryzae or Aspergillus niger (generalprocedure)

100 ml of YPD (Sherman et al., Methods in Yeast Genetics, Cold SpringHarbor Laboratory, 1981) is inoculated with spores of A. oryzae or A.niger and incubated with shaking at 37° C. for about 2 days. Themycelium is harvested by filtration through miracloth and washed with200 ml of 0.6M MgSO₄. The mycelium is suspended in 15 ml of 1.2M MgSO₄.10 mM NaH₂ PO₄, pH=5.8. The suspension is cooled on ice and 1 ml ofbuffer containing 120 mg of Novozym® 234, batch 1687 is added. After 5minutes 1 ml of 12 mg/ml BSA (Sigma type H25) is added and incubationwith gentle agitation continued for 1.5-2.5 hours at 37° C. until alarge number of protoplasts is visible in a sample inspected under themicroscope.

The suspension is filtered through miracloth, the filtrate transferredto a sterile tube and overlayered with 5 ml of 0.6M sorbitol, 100 mMTris-HCl, pH=7.0. Centrifugation is performed for 15 minutes at 100 gand the protoplasts are collected from the top of the MgSO₄ cushion. 2volumes of STC (1.2M sorbitol, 10 mM Tris-HCl, pH=7.5. 10 mM CaCl₂) areadded to the protoplast suspension and the mixture is centrifugated for5 minutes at 1000 g. The protoplast pellet is resuspended in 3 ml of STCand repelleted. This is repeated. Finally the protoplasts areresuspended in 0.2-1 ml of STC.

100 μl of protoplast suspension is mixed with 5-25 μg of the appropriateDNA in 10 μl of STC. Protoplasts are mixed with p3SR2 (an A. nidulansamdS gene carrying plasmid). The mixture is left at room temperature for25 minutes. 0.2 ml of 60% PEG 4000 (BDH 29576). 10 mM CaCl₂ and 10 mMTris-HCl, pH=7.5 is added and carefully mixed (twice) and finally 0.85ml of the same solution is added and carefully mixed. The mixture isleft at room temperature for 25 minutes, spun at 2500 g for 15 minutesand the pellet is resuspended in 2 ml of 1.2M sorbitol. After one moresedimentation the protoplasts are spread on the appropriate plates.Protoplasts are spread on minimal plates (Cove Biochem.Biophys.Acta 113(1966) 51-56) containing 1.0M sucrose, pH=7.0, 10 mM acetamide asnitrogen source and 20 mM CsCl to inhibit background growth. Afterincubation for 4-7 days at 37° C. spores are picked and spread forsingle colonies. This procedure is repeated and spores of a singlecolony after the second reisolation is stored as a defined transformant.

Immunological cross-reactivity: Antibodies to be used in determiningimmunological cross-reactivity may be prepared by use of a purifiedprotease. More specifically, antiserum against a protease of theinvention may be raised by immunizing rabbits (or other rodents)according to the procedure described by N. Axelsen et al. in: A Manualof Quantitative Immunoelectrophoresis, Blackwell ScientificPublications, 1973, Chapter 23, or A. Johnstone and R. Thorpe,Immunochemistry in Practice, Blackwell Scientific Publications, 1982(more specifically pp. 27-31). Purified immunoglobulins may be obtainedfrom the antisera, for example by salt precipitation ((NH₄)₂ SO₄),followed by dialysis and ion exchange chromatography, e.g. onDEAE-Sephadex. Immunochemical characterization of proteins may be doneeither by Outcherlony double-diffusion analysis (O. Ouchterlony in:Handbook of Experimental Immunology (D. M. Weir, Ed.), BlackwellScientific Publications, 1967, pp. 655-706), by crossedimmunoelectrophoresis (N. Axelsen et al., supra, Chapters 3 and 4), orby rocket immunoelectrophoresis (N. Axelsen et al., Chapter 2).

Media:

YPD: 10 g yeast extract, 20 g peptone, H₂ O to 810 ml. Autoclaved, 90 ml20% glucose (sterile filtered) added.

10×Basal salt: 66.8 g yeast nitrogen base, 100 g succinic acid, 60 gNaOH, H₂ O ad 1000 ml, sterile filtered.

SC-URA: 90 ml 10×Basal salt, 22.5 ml 20% casamino acids, 9 ml 1%tryptophan, H₂ O ad 806 ml, autoclaved, 3.6 ml 5% threonine and 90 ml20% glucose or 20% galactose added.

SC-H broth: 7.5 g/l yeast nitrogen base without amino acids, 11.3 g/lsuccinic acid, 6.8 g/l NaOH, 5.6 g/l casamino acids without vitamins,0.1 g/l tryptophan. Autoclaved for 20 min. at 121° C. After autoclaving,10 ml of a 30% galactose solution, 5 ml of a 30% glucose solution and0.4 ml of a 5% threonine solution were added per 100 ml medium.

SC-H agar: 7.5 g/l yeast nitrogen base without amino acids, 11.3 g/lsuccinic acid, 6.8 g/l NaOH, 5.6 g/l casamino acids without vitamins,0.1 g/l tryptophan, and 20 g/l agar (Bacto). Autoclaved for 20 min. at121° C. After autoclaving, 55 ml of a 22% galactose solution and 1.8 mlof a 5% threonine solution were added per 450 ml agar.

YNB-1 agar: 3.3 g/l KH₂ PO₄, 16.7 g/l agar, pH adjusted to 7.

Autoclaved for 20 min. at 121° C. After autoclaving, 25 ml of a 13.6%yeast nitrogen base without amino acids, 25 ml of a 40% glucosesolution, 1.5 ml of a 1% L-leucine solution and 1.5 ml of a 1% histidinesolution were added per 450 ml agar.

YNB-1 broth: Composition as YNB-1 agar, but without the agar.

FG-4-Agar: 35 g/L agar, 30 g/L Soy bean meal, 15 g/L maltodextrin(Glucidex 6), 5 g/L Bacto pepton, pH 7. Autoclaved 40 min at 121° C.

FG-4 medium: 30 g/L Soy bean meal, 15 g/L maltodextrin (Glucidex 6), 5g/L Bacto peptone. Autoclaved 40 min at 121° C.

MDU-2 medium: 45 g/L maltose, 1 g/L MgSO₄ -7 H₂ O, 1 g/L NaCl, 2 g/L K₂SO₄, 12 g/L KH₂ PO₄, 0.1 ml/L Pluronic 61 L, 0.5 ml/L Trace metalsolution. pH 5.0. Autoclaved 20 min at 121° C. 15 ml/L 50% sterilefiltered urea is added after autoclaving.

Casein overlayer gel: 1% agarose, 0.5% casein in a buffer with a pH of5.5. The gel was boiled and then cooled to 55° C. before the overlayerwas poured onto agar plates.

Fed batch fermentation

The medium used for fed-batch fermentation of protease I or II by A.oryzae comprised maltodextrin as a carbon source, urea as a nitrogensource and yeast extract.

The fed batch fermentation was performed by innoculating a shake flaskculture of the A. oryzae host cells in question into a medium comprising3.5% of the carbon source and 0.5% of the nitrogen source. After 24hours of cultivation at pH 5.0 and 34° C. the continuous supply ofadditional carbon and nitrogen sources were initiated. The carbon sourcewas kept as the limiting factor and it was secured that oxygen waspresent in excess. The fed batch cultivation was continued for 4 days,after which the enzymes could be recovered.

Characterization of enzymes

Proteolytic activity

1 hemoglobin protease unit (hpu) is defined as the amount of enzymeliberating 1 millimole of primary amino groups (determined by comparisonwith a serine standard) per minute under standard conditions asdescribed below:

A 2% (w/v) solution of hemoglobin (bovine, supplied by Sigma) isprepared with the Universal Buffer described by Britton and Robinson, J.Chem. Soc., 1931, p. 1451), adjusted to a pH of 5.5. 2 ml of thesubstrate solution are pre-incubated in a water bath for 10 min. at 25°C. 1 ml of an enzyme solution containing b g/ml of the enzymepreparation, corresponding to about 0.2-0.3 hpu/ml of the UniversalBuffer (pH 5.5) is added. After 30 min. of incubation at 25° C., thereaction is terminated by the addition of a quenching agent (5 ml of asolution containing 17.9 g of trichloroacetic acid, 29.9 g of sodiumacetate and 19.8 g of acetic acid made up to 500 ml with deionizedwater). A blank is prepared in the same way as the test solution withthe exception that the quenching agent is added prior to the enzymesolution. The reaction mixtures are kept for 20 min. in a water bathafter which they are filtered through Whatman 42 paper filters.

Primary amino groups are determined by their colour development witho-phthaldialdehyde (OPA), as follows: 7.62 g of disodium tetraboratedecahydrate and 2.0 g of sodium dodecylsulfate are dissolved in 150 mlof water. 160 mg of OPA dissolved in 4 ml of methanol were then addedtogether with 400 μl of β-mercaptoethanol after which the solution ismade up to 200 ml with water. To 3 ml of the OPA reagent are added 400μl of the filtrates obtained above, with mixing. The optical density(OD) at 340 nm is measured after about 5 min. The OPA test is alsoperformed with a serine standard containing 10 mg of serine in 100 ml ofUniversal Buffer (pH 5.5). The buffer alone is used as a blank. Theprotease activity is calculated from the OD measurements by means of thefollowing formula: ##EQU1## wherein OD_(t), OD_(b), OD_(ser) and OD_(B)is the optical density of the test solution, blank, serine standard andbuffer, respectively, C_(ser) is the concentration of serine (mg/ml) inthe standard (in this case 0.1 mg/ml), and MW_(ser) is the molecularweight of serine (105.09). Q is the dilution factor for the enzymesolution (in this case 8) and t_(i) is the incubation time in minutes(in this case 30 minutes).

Inhibition

The following inhibitors were tested:

Pepstatin (aspartic acid inhibitor) (1 mM)

PMSF (serine protease inhibitor) (0.1%)

PEFABLOC (serine protease inhibitor) (0.1%)

EDTA (metallo protease inhibitor) (0.1M)

all available from Sigma except for PEFABLOC which is available fromPentapharm, Basel, Switzerland.

Residual activity was determined as HPU/l at pH 5.5.

pH activity profiles were determined as HPU/l at different pH values(4-8).

pH-stabilitv was determined by letting an enzyme solution (0.3 HPU/l)stand for 30, 60 and 120 minutes, respectively, at 50° C. and differentpH-values (4-5-6-7-8) and measure proteolytic activity before and afterstanding.

Temperature activity profiles were determined as HPU/1 at differenttemperatures (15°-70° C.).

Temperature stability was determined by letting an enzyme solution (0.3HPU/1) stand for 30, 60 and 120 minutes at pH 5 and at differenttemperatures (25°-40°-50°-60° C.), and measure proteolytic activitybefore and after standing.

SDS gel electrophoresis and isoelectric focusing was carried out on thePhast-System from Pharmacia using a Gradient 8-25 and the IEF 3-9,respectively, according to the manufacturers instructions.

Specificity

The specificity of proteases of the invention is determined as follows:

0.5 ml of 1 mg/ml human insulin or bovine glucagon in Universal Buffer,pH 5.5 (vide supra), and 75 μl of protease I and II, respectively, (0.6hpu/l) in the same buffer were incubated for 120 min. at 37° C. Thereaction was terminated by adding 50 μl 1N hydrochloric acid.

The insulin or glucagon molecule was cleaved into a number of peptidefragments. These were separated and isolated by reverse phase HPLC usinga suitable C-18 column (Hibar LiChrosorb RP-18, 5 μm particles providedby Merck AG, Darmstadt, FRG). The fragments were eluted with thefollowing solvents:

A. 0.2M sodium sulfate and 0.1M phosphoric acid, pH 2.5;

B. Acetonitrile/water, 50%;

on a linear gradient of from 90% A/10% B to 80% A/20% B for 0-5 min. andsubsequently for 50 min. with 80% A/20% B. The isolated fragments weresubjected to amino acid sequencing by automated Edman degradation, usingan Applied Biosystems (Foster City, Calif., USA) Model 470A gas-phasesequencer, and the phenylthiohydantoin (PTH-) amino acids were analyzedby high performance liquid chromatography as described by L. Thim etal., "Secretion of human insulin by a transformed yeast cell", FEBSLetters 212(2), 1987, p.307, whereby the cleavage sites in the insulinor glucagon molecule were identified.

EXAMPLE 1

A library from A. aculeatus consisting of approx. 1.5×10⁶ individualclones in 150 pools was constructed.

DNA was isolated from 20 individual clones from the library andsubjected to analysis for cDNA insertion. The insertion frequency wasfound to be >90% and the average insert size was approximately 1400 bp.

DNA from some of the pools was transformed into yeast, and 50-100 platescontaining 200-500 yeast colonies were obtained from each pool. After3-5 days of growth, the agar plates were replica plated onto severalsets of agar plates. One set of plates was then incubated for 2-4 daysat 30° C. and overlayered with a casein overlayer gel for detection ofprotease activity. After incubation overnight at 30° C.,protease-positive colonies were identified as colonies surrounded by awhite halo.

Cells from enzyme-positive colonies were spread for single colonyisolation on agar, and an enzyme-producing single colony was selectedfor each of the protease-producing colonies identified.

The positive clones were obtained as single colonies, the cDNA insertswere amplified directly from the yeast colony using biotinylatedpolylinker primers, purified by magnetic beads (Dynabead M-280, Dynal)system and characterized individually by sequencing the 5'-end of eachcDNA clone using the chain-termination method (Sanger et al., 1977) andthe Sequenase system (United States Biochemical). The DNA sequences oftwo enzyme genes are shown in SEQ ID Nos. 1 and 2, respectively.

Subsequently, the cDNA encoding the protease was isolated for expressionin Aspergillus as described above and transformed into E. coli usingstandard procedures. Two E. coli colonies were isolated from each of thetransformations and analysed with the restriction enzymes HindIII andXbaI which excised the DNA insert. DNA from one of these clones wasretransformed into yeast strain JG169.

The DNA sequences of several of the positive clones were determined. TwoDNA sequences encoding a protease are shown in SEQ ID Nos. 1 and 2,respectively.

EXAMPLE 2

In order to express the genes in Aspergillus, cDNA is isolated from oneor more representatives of each family using the above describedprocedure by digestion with HindIII/XbaI or other appropriaterestriction enzymes, size fractionation on a gel and purification andsubsequently ligated to pHD414, resulting in the plasmids pA1P1 andpA1P2. After amplification in E. coli, the plasmids are transformed intoA. oryzae or A. niger according to the general procedure describedabove.

Test of A. oryzae transformants

Each of the transformants was inoculated in the center of a Petri dishwith FG-4 agar. After 5 days of incubation at 30° C. 4 mm diameter plugswere removed from the center of the colonies by means of a corkscrew.The plugs were embedded in a casein overlayer gel, containing 0.5%casein and 1% agarose in a buffer with a pH of 5.5, and incubatedovernight at 40° C. The protease activity was identified as describedabove. Some of the transformants had halos which were significantlylarger than the Aspergillus oryzae background. This demonstratesefficient expression of protease in Aspergillus oryzae. The 8transformants with the highest protease activity were selected andinoculated and maintained on YPG-agar.

Each of the 8 selected transformants were inoculated from YPG-agarslants on 500 ml shake flask with FG-4 and MDU-2 media. After 3-5 daysof fermentation with sufficient agitation to ensure good aeration, theculture broths were centrifuged for 10 minutes at 2000 g and thesupernatants were analyzed.

A volume of 15 μl of each supernatant was applied to 4 mm diameter holespunched out in a casein overlayer gel (25 ml in a 13 cm diameter Petridish). The protease activity was identified by the formation of a whitehalo on incubation.

Fed batch fermentation

Subsequently, protease I and II, respectively, were produced by fedbatch fermentation of A. oryzae expressing the enzyme using theprocedure described above.

EXAMPLE 3

Characterization of Protease I and II

The supernatant resulting from the fed batch fermentation above was usedfor the characterization performed as described in the Materials andMethods section above. Proteolytic activity was measured as HPU/l at pH5.5 using the above described procedure.

Inhibition tests gave the following results:

    ______________________________________                                                % Residual activity                                                           EDTA  Pepstatin  PEFABLOC  PMSF                                       ______________________________________                                        Protease I                                                                              104     91         83       92                                      Protease II                                                                              97      9         90      108                                      ______________________________________                                    

The inhibition of Protease II by Pepstatin shows that it is an asparticprotease of the Pepsin type. Protease I is not inhibited by Pepstatinand is therefore not positively identified as an aspartic protease.Optimum activity at pH 5, on the other hand, shows that it is an acidprotease. pH activity profiles are shown in FIG. 1. It is seen that bothenzymes have optimum activity at pH 5, and that Protease I is active ina more narrow range than Protease II. Thus, protease I exhibits morethan 60% activity in the range of pH 4-6, whereas Protease II exhibitsmore than 60% activity in the range of pH 4-7.

By SDS gel analysis the molecular weights of Protease I and II,respectively, were estimated to 23.000 and 37.000 kDa, respectively.From the IEF analysis the pI of both enzymes are estimated to about 4.

FIG. 2 shows the temperature-activity profiles. They are rather similarfor the two enzymes, but with slightly different optimum temperatures,50° C. for Protease I and 45° C. for Protease II.

FIGS. 3 and 4 show the pH-stability. For each pH, the zero-timeactivities have been set to 100%, and the absolute values obtained aretherefore different. Both proteases are stable at pH 4 and 5. At pH 6,Protease I is unstable, while Protease II has a certain stability (40%residual activity after 30 min). Both enzymes are unstable at pH 7.

FIGS. 5 and 6 show the temperature-stability. For each temperature, thezero-time activities have been set to 100%, and the absolute valuesobtained are different. Both proteases are stable up to 50° C., butunstable at 60° C.

Based on the results obtained on hydrolysis of insulin and glucagon byProtease I and Protease II it was found that Protease II does not reactwith insulin, whereas both proteases hydrolyse glucagon. It can beconcluded, that Protease I is a rather unspecific protease, whileProtease II is more specific. It was found that protease II is capableof cleaving the Lys-Tyr and the Phe-Val bonds found in bovine glucagon(the sequence of which is shown in Bromer, Sinn, and Behrens, J. Amer.Chem. Soc., Vol. 79, p. 2807, 1958).

EXAMPLE 4

Use of a protease of the invention for viscosity reduction

Soy flour (prepared from defatted and peeled soy beans) were pelletizedat 95° C. and grinded afterwards. The soy flour is suspended indeionized water to 15% dry substance. 5 mg protease I enzyme protein perg of dry substance and 5 mg protease II enzyme protein per g of drysubstance, respectively, was added to the soy slurry. The slurry wasincubated at 40° C. and pH 5-6. The viscosity in the slurry was measuredafter 1, 2 and 24 hours of incubation on a Brookfield LV DV IIIviscometer using a small sample adaptor with spindle #31 at 250 rpm. Theresidual viscosities were as follows:

    ______________________________________                                                      Prot. I                                                                             Prot. II                                                  ______________________________________                                        1 hour          73%     47%                                                   2 hours         59%     38%                                                   ______________________________________                                    

EXAMPLE 5

Use of a protease of the invention for cleaning of contact lenses

In the field of contact lens cleaning it is essential to regularly haveboth an efficient disinfection and cleaning of the contact lens. One ofthe most effective ways of disinfecting contact lenses is to immersethem into a solution containing 3% H₂ O₂ at pH 3.5 for at least 20minutes. The H₂ O₂ is neutralized with e.g. catalase or a platinum discbefore inserting the lens into the eye. Unfortunately no commerciallyinteresting protease till date has been shown to have good effect underthese harsh conditions, so a cumbersome second step with addition of aprotease after H₂ O₂ -neutralization is needed to remove the proteindeposits on the contact lens. Porcine pepsin is superior to thepresently used serin protease (Subtilisin carlsberg) but is troublesomebecause of the viruses often associated with mammal products.

Protease I and II, respectively, of the invention have been tested withrespect to the ability to remove denatured protein from a contact lens.They have been compared to the presently used serin protease and also toporcine pepsin, although the latter is interesting from a technicalperspective rather than a commercial perspective.

The experimental protocol was as follows:

Materials: Hen Lysozyme, L-6878 from Sigma "Rythmic" contact lens' fromEssilor (Type II lens, high-water, nonionic) Protease I produced asdescribed above Protease II produced as described above Porcine pepsin,P-6887 from Sigma Subtilisin carlsberg, Clear-Lens Pro® (Novo NordiskA/S).

Standard buffer: 0.05M Na₂ HPO₄, 0.9% NaCl pH 7.5

Reagent buffer: 0.05M Na₂ HPO₄, 0.9% NaCl, 3% H₂ O₂, pH 3.5

Scintillation liquid, Optiphase "HiSafe III"

Hen lysozyme from Sigma was labelled with ¹⁴ C through reductivemethylation and purified.

A solution was made containing 0.05M Na₂ HPO₄, 0.9% NaCl and 0.2 mg/mllysozyme pH 7.5. An amount of ¹⁴ C-labelled lysozyme was added so theCPM (Counts Per Minute) is approximately 200.000. 1.0 ml of the solutionwas transferred to a scintillation glass. The contact lens was added andthe glass placed in a water-bath at 85° C. for 30 minutes.

The contact lens was then rinsed in 3×3 ml reagent buffer. It wasquartered with a scalpel. Each quarter was transferred to a newscintillation glass containing 3 ml of the reagent buffer.

Different amounts of the protease to be tested were added so the finalconcentrations were 0.1, 0.5, 2.5, 5, 12.5, 50 and 200 μg enzymeprotein/ml reagent buffer.

The reaction took place over four hours at 25° C. The quarter lenseswere rinsed in 2×3 ml standard buffer. 12 ml scintillation liquid wasadded and CPM was measured in a Packard 2500 TR liquid scintillationcounter.

Four lenses were needed to evaluate each protease: Double determinationswere made over two days, and a blind reference was needed for each lens.

The relative amount of lysozyme removed from the lens during thecombined disinfection/cleaning was calculated from the mass balance ofeach quarter lens. FIG. 7 gives a graphic presentation of theperformance of the different proteases.

Both proteases are highly suitable for contact lens cleaning purposes.Protease I was found to be very superior to the other proteases.Protease II is very close to porcine pepsin in performance whereas thepresently used serin protease shows poor performance. A furtheradvantage of the acidic proteases of the invention is the low activityat the neutral pH found in tear fluid. This lowers the risk ofirritation if the lenses are not rinsed properly afterdisinfection/cleaning.

EXAMPLE 6

Use of a protease of the invention for baking

Procedure:

To 10 grams of cake flour 5.9 g of water are added. The water containsdifferent concentrations of Protease I, Protease II and Neutrase®(available from Novo Nordisk A/S). The dough is mixed on a Glutamic 2200mixer for one minut. It is then placed in a plastic bag and is incubatedfor 25 min. at 32° C.

Thereafter the dough is washed with 2% NaCl (aqeuous solution) using theGlutamic mixer in order to remove starch and leave gluten in the dough.

The gluten lump is then rolled by hand untill homogeneity and is pressedinto the shape of a cylinder which is about 0.5 cm high and 2.5 cm indiameter and has a hole in the center. The gluten lump is pressed intoshape for 30 min at 25° C. Subsequently, the gluten cylinder is hung ona hook and a 2 g weight is placed in the hole. Everything is placedunder water at 25° C.

The stretching of the gluten cylinder is thereafter measured every 15min untill it breaks.

Enzymes are dosed on an Anson Unit basis (AU), initially trying with 7.5mAU/kg flour, which is the optimal dose for Neutrase®. (In theAnson-Hemoglobin method for the determination of porteolytic activitydenatured hemoglobin is digested at a temperature of 25° C., pH 7.5 anda rection time of 10 min. The undigested hemoglobin is precipitated withtrichloroacetic acid (TCA) and the amount of TCA soluble product isdetermined with phenol reagent, which gives a blue colour with tyrosineand tryptophan. 1 AU is the amount of enzyme which digests hemoglobin atan initial rate such that there is liberated per minute an amount of TCAsoluble product which gives the same colour with phenol reagent as onemilliequivalent of tyrosine).

FIG. 8 shows the stretching curves of gluten without enzyme and withNeutrase®, Protease I and Protease II. The curves are means of 6-7determinations with the same dose of enzyme. As can be seen, theaddition of all 3 proteases led to a faster stretching of the gluten.

Protease I in a dose of 7.5 mAU/kg flour does not weaken the gluten asmuch as the same dose of Neutrase®. The gluten cylinder gets longerbefore it breaks, and the rate of elongation is lower.

Addition of 2.3 mAU/kg flour of protease II, which was the largestamount possible in this system, almost weakens the gluten as much as 7.5mAU/kg of Neutrase®. The gluten breaks a little later, and the shape ofthe curves are not identical. This shows that protease II isapproximately 3 times as efficient on an AU basis as Neutrase® forgluten weakening.

In conclusion the proteases of the invention constitutes a desirablealternative to chemicals conventionally used for gluten weakening, awidely used example of which is SMS (sodium metabisulphite).

EXAMPLE 7

Use of a protease of the invention for animal feed

Ground defatted feed quality soy was mixed with deionised water underthe conditions described below. The hydrolysis was carried out in twosteps in order to simulate the pH conditions in the stomach and thesmall intestine. The performance of Protease II of the invention wascompared with that of Bio-Feed Pro, which by Brenes et al., 1993, hasbeen demonstrated to result in improved weight gain and feed efficiencywhen used in broiler diets.

    ______________________________________                                        Hydrolysis conditions:                                                        ______________________________________                                        Hydrolysis mixture                                                                           70 g ground defatted soy                                                      330 g deionised water                                          Temperature    40° C.                                                  pH             1st step 4.0                                                                  2nd step 6.5                                                   Time           1st step 180 minutes                                                          2nd step 180 minutes                                           Enzymes        1st step I) Pepsin 1.92 g                                                              (Merck art. 7190)                                                             II) I + Bio-Feed Pro 3.0 L                                                    5.5 AU/kg soya                                                                III) I + protease II                                                          0.19 AU/kg soya                                                      2nd step I) Pancreatin 6 g                                                             (Sigma P 1750)                                                                II) I + Bio-Feed Pro 3.0 L                                                    5.5 AU/kg soya                                                                III) I + Protease II                                                          0.19 AU/kg soya                                       ______________________________________                                    

Bio-Feed Pro® is available from Novo Nordisk A/S. Protease II wasobtained as described above.

The enzymes were added at start 0 minutes. During the hydrolysis ° Brixand osmolality were measured to follow the reaction course. According toAdler-Nissen (1986) the osmolality values can be used for calculation ofthe Degree of Hydrolysis (DH) by the following equation: ##EQU2## WhereΔ is the increase in osmolality mOSM, S % is protein concentration, ω isthe calibration factor for the osmometer, h_(tot) is the total number ofpeptide bonds in the protein substrate (meqv/g protein), and f_(osm) isthe factor for converting % to g/kg H₂ O. ##EQU3##

Further the average Molecular Weight was analysed of the sulphosalicylic acid soluble phase of the N-components of the hydrolysismixture after 90 minutes (1st step) and 0, 15 and 180 minutes (2ndstep). The Molecular Weight analyses were performed by the followingmethod:

1. Principle

The sample is diluted, filtrated and injected into a liquidchromatographic system, operating in the Gel Permeation Chromatography(GPC) mode. This separation technique utilizes a liquid flow through acolumn filled with porous particles having pores with a well-definedpore diameter. When a solution of peptides having different molecularsize passes through the column, the small peptides will be able to flowinto the pores while the larger peptides will be excluded from thepores. Thus, the peptides in a solution will be separated according tomolecular size (and weight) as the large peptides will be eluted fasterfrom the column than the small peptides. A detector at the column outletcontinuously measures the effluent. The chromatographic system iscalibrated with peptides with known molecular weight.

2. Chromatographic equipment

2.1 HPLC system consisting of High Pressure pump, WATERS M 510, flowrate 0.7 ml/min. Injector, Waters WISP M 710 Detector, Waters M 440,with wavelength extension to 214 nm.

2.2 GPC column, 3×TSK G 2000 SWXL, 7.8 mm×300 mm, connected in seriesand operated at ambient temperature.

2.3 Integration/data processing, Waters 820 MAXIMA SIM chromatographydata system with 810/820 GPC option.

3. Reagents

3.1 Phosphate buffer, NaH₂ PO₄ 2H₂ O

3.2 Ammoniumchloride, NH₄ CL

3.3 Trifluoroacetic acid (TFA), CF₃ COOH

3.4 Acetonitrile, CH₃ CN

3.5 Mobile phase:

0.05M Phosphate buffer/0.5M Ammoniumchloride solution containing 0.1%TFA and 25% Acetonitrile

4. Description

4.1 Calibration

The chromatographic system is calibrated by means of injections ofnumerous peptide standards with known molecular weight. The molecularweight of each standard is plotted semilogarithmic versus the observedvolume of mobile phase needed to elute the peptide from the column. By aleast squares calculation the best fitting 3rd order polynomium iscalculated. This curve represents the calibration curve.

4.2 Analysis

The sample is diluted/dissolved in mobile phase to approx. 5 mg/ml. Thesolution is filtered through a 22 μm filter and 20 μl is used forinjection into the chromatograph. The detector response versus elutionvolume is recorded. The recorded curve--the chromatogram--shows theactual molecular weight distribution of the sample. To allow forcalculations as to accumulated weight distribution and average molecularweight calculations, the chromatogram is divided into small time (andelution volume) segments--each segment being characterized by theelution volume and the area of the chromatogram over the time interval.

5. Calculation

Results are given in terms of weight and number average molecularweights. ##EQU4## where

M_(w) : Weight average molecular weight

M_(n) : Number average molecular weight

A_(i) : Area of chromatogram for each segment, measured as theaccumulated detector response over each time interval.

M_(w),i : The corresponding molecular weight for each segment. The valueis calculated by means of the calibration curve using the averageelution volume over the time interval.

RESULTS

The values for ° Brix, mOSM and %DH are given in the table below:

    __________________________________________________________________________    HYDROLYSIS OF FEED SOY WITH PEPSIN, BIO-FEED PRO, PROTEASE II AND             PANCREATIN                                                                    __________________________________________________________________________    PARAMETER: °BRIX                                                               STEP I, MIN.                                                          ENZYME  0   5   10  15  30  45  60  90  120 180                               PEPSIN  5.09                                                                              6.135                                                                             6.505                                                                             6.625                                                                             6.9275                                                                            7.345                                                                             7.745                                                                             8.175                                                                             8.695                                                                             9.195                             BIO-FEED                                                                              5.09                                                                              6.125                                                                             6.325                                                                             6.545                                                                             7.245                                                                             7.465                                                                             7.565                                                                             8.185                                                                             8.545                                                                             9.105                             PRO                                                                           PROTEASE II                                                                           5.09                                                                              6.505                                                                             6.845                                                                             7.105                                                                             7.825                                                                             8.405                                                                             8.605                                                                             9.305                                                                             9.705                                                                             10.205                                    STEP II, MIN.                                                         ENZYME  180 185 190 195 210 225 240 270 300 360                               PEPSIN  12.965                                                                            15.135                                                                            15.4025                                                                           15.637                                                                            16.135                                                                            16.415                                                                            16.705                                                                            17.075                                                                            17.437                                                                            18.075                            BIO-FEED PRO                                                                          12.965                                                                            15.165                                                                            15.165                                                                            15.725                                                                            16.105                                                                            16.4                                                                              16.525                                                                            16.885                                                                            17.285                                                                            17.805                            PROTEASE II                                                                           13.605                                                                            15.605                                                                            15.925                                                                            16.025                                                                            16.365                                                                            16.685                                                                            16.885                                                                            17.105                                                                            17.505                                                                            18.145                            __________________________________________________________________________    PARAMETER: mOSM                                                                       STEP I, MIN.                                                          ENZYME  0   5   10  15  30  45  60  90  120 180                               PEPSIN  347 362 362 360.5                                                                             363.5                                                                             366 368 370.5                                                                             373 381.5                             BIO-FEED PRO                                                                          347 365 364 366 367 371 373 379 380 385                               PROTEASE II                                                                           347 371 382 391 411 421 427 439 447 463                                       STEP II, MIN.                                                         ENZYME  180 185 190 195 210 225 240 270 300 360                               PEPSIN  468 996 1009                                                                              1015.5                                                                            1037.5                                                                            1054.5                                                                            1068.5                                                                            1087.5                                                                            1111                                                                              1148                              BIO-FEED PRO                                                                          468 1013                                                                              1017                                                                              1033                                                                              1048                                                                              1058                                                                              1069                                                                              1094                                                                              1113                                                                              1145                              PROTEASE II                                                                           548 1118                                                                              1127                                                                              1135                                                                              1152                                                                              1161                                                                              1176                                                                              1201                                                                              1227                                                                              1284                              __________________________________________________________________________    PARAMETER: mOSM INCREASE                                                              STEP I, MIN.                                                          ENZYME  0   5   10  15  30  45  60  90  120 180                               PEPSIN  0   15  15  13.5                                                                              16.5                                                                              19  21  23.5                                                                              26  34.5                              BIO-FEED PRO                                                                          0   18  17  19  20  24  26  32  33  38                                PROTEASE II                                                                           0   24  35  44  64  74  80  92  100 116                                       STEP II, MIN.                                                         ENZYME  180 185 190 195 210 225 240 270 300 360                               PEPSIN  0   528 541 547.5                                                                             569.5                                                                             586.5                                                                             600.5                                                                             619.5                                                                             643 680                               BIO-FEED PRO                                                                          0   545 549 565 578 590 601 626 645 677                               PROTEASE II                                                                           0   570 579 587 604 613 628 653 679 736                               __________________________________________________________________________    PARAMETER: % DH                                                                       STEP I, MIN.                                                          ENZYME  0   5   10  15  30  45  60  90  120 180                               PEPSIN  0   2.86                                                                              2.86                                                                              2.57                                                                              3.14                                                                              3.62                                                                              4.00                                                                              4.47                                                                              4.95                                                                              6.57                              BIO-FEED PRO                                                                          0   3.43                                                                              3.24                                                                              3.62                                                                              3.81                                                                              4.57                                                                              4.95                                                                              6.09                                                                              6.28                                                                              7.24                              PROTEASE II                                                                           0   4.57                                                                              6.66                                                                              8.38                                                                              12.19                                                                             14.09                                                                             15.23                                                                             17.52                                                                             19.04                                                                             22.09                             __________________________________________________________________________

The results of the molecular weight analysis is given below:

    ______________________________________                                                 Step I                                                                              step II                                                                 90 min                                                                              0 min     15 min  180 min                                      ______________________________________                                        Pepsin     960     1070      680   520                                        Bio-Feed Pro                                                                             880     1020      690   510                                        Protease II                                                                              650      630      530   480                                        ______________________________________                                    

The apparent Molecular Weight is increased when pH is adjusted to 6.5 asundigested soy protein is more soluble at pH 6.5 than at pH 4.0.

It is seen that protease II releases more protein and peptides anddegrade the proteins more than Bio-Feed Pro, it therefore concluded thatprotease II is superior to Bio-Feed Pro.

Since protease II of the invention has optimum activity in acid pHrange, this enzyme will therefore perform already in the stomach of theanimal, thus an overall improvement on feed efficiency should beachieved compared to Bio-Feed Pro when applied in feed for younganimals. The above results supports this conclusion.

REFERENCES

Aviv, H. & Leder, P. 1972. Proc. Natl. Acad. Sci. U. S. A. 69:1408-1412.

Becker, D. M. & Guarante, L. 1991. Methods Enzymol. 194: 182-187.

Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J. & Rutter, W. J. 1979.Biochemistry 18: 5294-5299.

Gubler, U. & Hoffman, B. J. 1983. Gene 25: 263-269.

Sambrook, J., Fritsch, E. F. & Maniatis, T. 1989. Molecular Cloning: ALaboratory Manual. Cold Spring Harbor Lab., Cold Spring Harbor, N.Y.

Sanger, F., Nicklen, S. & Coulson, A. R. 1977. Proc. Natl. Acad. Sci.U.S.A. 74: 5463-5467.

Brenes, A. et al., Poultry Science, 72, 2281-2293, 1993.

Adler-Nissen, J. Enzymic Hydrolysis of Food Proteins, Elsevier AppliedScience Publishers London and New York, 1986.

Takahashi, K. et al., 1991, The Primary Structure of Aspergillus nigerAcid Proteinase A*, The Journal of Biol. Chemistry, Vol. 266, No. 29,pp. 19480-19483.

Choi, G. H. et al., Molecular analysis and overexpression of the gneeencoding endothiapepsin, an aspartic protease from Cryphonectriaparasitica, 1993, Gene 125: 135-131.

Gomi, K. et al., 1993, Cloning and Nucleotide Sequence of the AcidProtease-encoding Gene (pepA) from Aspergillus oryzae, Biosci. Biotech.Biochem., 57(7): 1095-1100.

Inoue, H. et al., 1991, The Gene and Deduced Protein Sequences of theZymogen of Aspergillus niger Acid Proteinase A*, The Journal ofBiological Chemistry, Vol. 266, No. 29, pp. 19484-19489.

Berka, R. M. et al., 1993, Isolation and characterization of theAspergillus oryzae gene encoding aspergillopepsin O, Gene, 125: 195-198.

Berka, R. M. et al., 1990, Molecular cloning and deletion of the geneencoding aspergillopepsin A from Aspergillus awamori, Gene 86: 153-162.

Berka, R. M. et al., 1990, Corrigendum, Molecular cloning and deletionof the gene encoding aspergillopepsin A from Aspergillus awamori, Gene96: 313.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 34                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1124 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Aspergillus aculeatus                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       AATTAAGCATCCTCCATCTTCAAAGCTCAATCTCGCTAACTCCCGCTCTTCTCTCGATCT60                CATCATCCCAATAACTCGGACACAATGAAGACCTCTGCTCTCTTGACCGCTGGCCTGTTG120               GCCACCGCTGCTATTGCTGCTCCTCTCACCGAGAAGCGCGCAGCTGCTCGCGCTGCCAAG180               CGTGGCACCAGCCGCAAGAGCAACCCCCCTCTCAAGCCCGGCACCAGCGAGGCCATCAAC240               CTGACCGGCTCCAAGAACACCGAGTACTCGTCCAACTGGGCCGGCGCCGTGCTCATCGGC300               ACCGGCTACACTGCCGTCACCGCCGAGTTCACCATTCCCACCCCCTCTCTCCCCTCCGGT360               GCCTCCAGCCGCGAGCAGTACTGTGCCTCCGCCTGGGTCGGTATCGACGGTGACACCTGC420               GACACCGCCATCCTGCAGACCGGTCTCGACTTCTGTATCGAGGGCAGCACCGTTAGCTAC480               GACGCCTGGTACGAGTGGTACCCCGACTATGCCTACGACTTCAGCGGCATCAGCTTCTCC540               GCCGGCGACGTTGTCAAGGTCACCGTCGACGCCACCAGCAAGACCGCCGGTACCGCCACC600               GTCGAGAACGTCACCAAGGGCACCACCGTCACCCACACCTTCAGCGGTGGTGTTGATGGT660               GATCTCTGCGAGTACAACGCCGAGTGGATCGTCGAGGACTTCGAGGAGAACTCCTCCCTC720               GTCCCCTTCGCCGACTTCGGCACCGTCACCTTCTCCAGCGCCTACGCCACCAAGAGCGGC780               TCCACCGTTGGTCCCTCCGGCGCCACCATCATCGACATCGAGCAGAACAACAAGGTTCTC840               ACCTCCGTCTCGACCTCCAGCAGCTCCGTCACCGTCGAGTATGTTTGAAGGGGACTCCTG900               GGGATGTGAAGCGAGAATGCGGCTTGGGTGGTTGGAGGTCCTTTGGGACGTCGAACGCCT960               AGGATTCAACGGGATGAGATCATTGGAAATGAAGACGAGAATGAGCGAATACTGTCACTG1020              ATTGAGATTGTGCTTTGTTGATTGTGTTAGGGGCTTGCCTCTGAAAATTGAGCTTAGTGT1080              TGCTGCAATATGATTGCTGTGGTTGAGAAAAAAAAAAAAAAAAA1124                              (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1425 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Aspergillus aculeatus                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       CTGCTTCTCCTTCTCTTCCTCCTCGTGATATCTGCTTGAACATCTCCTCATCATGGTCGT60                CCTCAACAAGGCTGCAGCCCTTCTTCTGGGTCTGACCACCGCCGCCACTGCGGCTCCCCT120               GGCCGAGAAGCAGGCTTCTGTCCCGGTCAAGAACTTCTCCGTCAAGCAGGTCGAGAAGGA180               GGGCAGCAAGGGACGTACCGTTAACCTGCCGGGTCTGTATGCGAATGCGCTGGCCAAGTA240               TGGCGCCCAGGTGCCGGCCAGCGTCAAGGCCGCCGCCGTCAGTGGCAGCGTCGTGACCAC300               CCCGCAGGCCAACGACGTCTCCTACCTGACCCCCGTCACCGTGGGCAGCTCGACCTTGAA360               CCTGGACTTCGACACCGGATCCGCCGATCTCTGGGTCTTCTCCTCGGAGCTGGCCGCCTC420               CTCGCGCACCGGCCACAGCATCTACACCCCCGGCAGCACCGCCCAGAAGCTGTCCGGCTA480               CAGCTGGAGCATCTCCTACGGCGACGGCAGCTCCGCCAGCGGCGACGTCTACAAGGACAA540               GGTCACCGTCGGCACGGTGACGGCCAGCAGCCAGGCCGTCGAGGCCGCCAGCCGCATCAG600               CTCCGAGTTCGTCCAGGACACCGACACCGACGGTCTGTTGGGTCTGGCCTTCAGCTCGAT660               CAACACGGTCTCCCCCCGGGCCCAGACCACCTTCTTCGACACCGTCAAGTCCAGCCTGGA720               CAGCCCCCTCTTCGCCGTCGACCTGAAGTACCACGCCGCCGGTACCTACGATTTCGGGTT780               CATCGACTCCTCCAAGTACACCGGCTCCCTGACCTACGCCAACGTCGACGACTCCCAGGG840               CTTCTGGCAATTCACCGCCAGCGGCTACAGCGTGGGCTCGGCCTCCCACTCCTCCTCTTT900               CTCCGCCATTGATGACACCGGCACCACCCTCATCCTCCTCGACGACTCCATCGTCTCCAC960               CTACTACAAGAGCGTCAGCGGCGCCTCCTACAGCTACAACTACGGCGGCTACGTCTTCTC1020              CTGCTCCGCCAGCCTGTCCAACTTCAGCGTCAAGATCGGCTCCTACACCGCCGTCGTCCC1080              CGGCAAGTACATCAACTACGCCCCCATCTCCACCGGCAGCTCCACCTGCTACGGCGGCAT1140              CCAGTCCAACGAGGGCCTCGGTCTGTCCATCCTGGGTGATGTCTTCCTCAAGAGCCAGCA1200              CGTGGTCTTTGACTCGCAGGGTCCGAGAATCGGGTTCGCCGCGCAGGCCTAGATCGTTTG1260              ATTGGGGTTGTGGATGTGGGTGATGCTTGGTGGTGGTCTGAGTCGTGGTCTATGTGGGCG1320              TGAATATAGTACTGTATATAGTACTGTACATAGGGGGGTGGTGAACATATGGTCTGGTCG1380              ATGAATATATGTCTTTGATGTTATGCTTCTGTGGAAAAAAAAAAA1425                             (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Aspergillus aculeatus                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       AATTAAGCATCCTCCATCTT20                                                        (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       CAAAGCTCAATCTCGCTAAC20                                                        (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       TCCCGCTCTTCTCTCGATCT20                                                        (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Aspergillus aculeatus                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       CATCATCCCAATAACTCGGA20                                                        (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       CAAAATGAAGACCTCTGCTC20                                                        (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Aspergillus aculeatus                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       TCTTGACCGCTGGCCTGTTG20                                                        (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Aspergillus aculeatus                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       GCACCGCTGCTATTGCTGCT20                                                        (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Aspergillus aculeatus                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      CCTCTCACCGCGAAGCGCGC20                                                        (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Aspergillus aculeatus                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      ACGTGCTCGCGCTGCCAAGC20                                                        (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Aspergillus aculeatus                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      TGGCACCAGCCGCAAGAGCA20                                                        (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Aspergillus aculeatus                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      AGGGGGGTCTCAAGCCCGGC20                                                        (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Aspergillus aculeatus                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      ACCCAGCGAGGCCATAACCT20                                                        (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Aspergillus aculeatus                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      GACCGGCTCCAAGAACACCG20                                                        (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Aspergillus aculeatus                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      GAGGTACTCGTCCAACTGGG20                                                        (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Aspergillus aculeatus                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      CCGGCGCCGTGCCAT15                                                             (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 295 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Aspergillus aculeatus                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      AATTAAGCATCCTCCATCTTCAAAGCTCAATCTCGCTAACTCCCGCTCTTCTCTCGATCT60                CATCATCCCAATAACTCGGACAAAATGAAGACCTCTGCTCTCTTGACCGCTGGCCTGTTG120               GCACCGCTGCTATTGCTGCTCCTCTCACCGCGAAGCGCGCACGTGCTCGCGCTGCCAAGC180               TGGCACCAGCCGCAAGAGCAAGGGGGGTCTCAAGCCCGGCACCCAGCGAGGCCATAACCT240               GACCGGCTCCAAGAACACCGGAGGTACTCGTCCAACTGGGCCGGCGCCGTGCCAT295                    (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Aspergillus aculeatus                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      CTGCTTCTCCTTCTCTTCCT20                                                        (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Aspergillus aculeatus                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      CCTCGTGATATCTGCTTGAA20                                                        (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Aspergillus aculeatus                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      CATCTCCTCATCATGGTCGT20                                                        (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Aspergillus aculeatus                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      CCTCAACAAGGTGCAGCCTT20                                                        (2) INFORMATION FOR SEQ ID NO:23:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                      CTTCTGGGTCTGACCACCGC20                                                        (2) INFORMATION FOR SEQ ID NO:24:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Aspergillus aculeatus                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                      CGCCACTGGTCCCCTGGCCG20                                                        (2) INFORMATION FOR SEQ ID NO:25:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Aspergillus aculeatus                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                      AGCCGCAGGCTTCTGTCCGG20                                                        (2) INFORMATION FOR SEQ ID NO:26:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Aspergillus aculeatus                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                      TCAAGAACTTCTCCGTCAAG20                                                        (2) INFORMATION FOR SEQ ID NO:27:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Aspergillus aculeatus                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                      CAGGTCGAGAAGGCGGGCAG20                                                        (2) INFORMATION FOR SEQ ID NO:28:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Aspergillus aculeatus                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                      CAAGGGACGTACCGTTAACC20                                                        (2) INFORMATION FOR SEQ ID NO:29:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Aspergillus aculeatus                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                      TGCCGGGTCTGTATGCGAAT20                                                        (2) INFORMATION FOR SEQ ID NO:30:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Aspergillus aculeatus                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                      GCGCTGGCCAAGTATGGCGC20                                                        (2) INFORMATION FOR SEQ ID NO:31:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Aspergillus aculeatus                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                      CCAGGTGCGGCCAGCGTCAA20                                                        (2) INFORMATION FOR SEQ ID NO:32:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Aspergillus aculeatus                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                      GGCCGCCGCCGTCAGTGGCA20                                                        (2) INFORMATION FOR SEQ ID NO:33:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Aspergillus aculeatus                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                      GCGTCGTGACCACCCGCAGGCCAACGACG29                                               (2) INFORMATION FOR SEQ ID NO:34:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 309 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Aspergillus aculeatus                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                      CTGCTTCTCCTTCTCTTCCTCCTCGTGATATCTGCTTGAACATCTCCTCATCATGGTCGT60                CCTCAACAAGGTGCAGCCTTCTTCTGGGTCTGACCACCGCCGCCACTGGTCCCCTGGCCG120               AGCCGCAGGCTTCTGTCCGGTCAAGAACTTCTCCGTCAAGCAGGTCGAGAAGGCGGGCAG180               CAAGGGACGTACCGTTAACCTGCCGGGTCTGTATGCGAATGCGCTGGCCAAGTATGGCGC240               CCAGGTGCGGCCAGCGTCAAGGCCGCCGCCGTCAGTGGCAGCGTCGTGACCACCCGCAGG300               CCAACGACG309                                                                  __________________________________________________________________________

We claim:
 1. An isolated and purified DNA sequence encoding an enzymehaving the same activity as an enzyme having a pH optimum between 4 and7, exhibits protease activity in 5% hydrogen peroxide, is obtainablefrom filamentous fungus, and is encoded by a DNA sequence depicted inSEQ ID NO: 1 or
 2. 2. The isolated and purified sequence of claim 1 inwhich said DNA sequence is obtainable from a strain of Aspergillus,Rhizopus, Trichoderma, Penicillium, Fusarium, Schytalidium or Humicola.3. The isolated and purified DNA sequence of claim 2, in which the DNAsequence is obtainable from a strain of Aspergillus.
 4. The isolated andpurified DNA sequence of claim 3, in wich the DNA sequence is obtainablefrom a strain of Aspergillus aculeatus, Aspergillus niger or Aspergillusoryzae.
 5. The isolated and purified DNA sequence of claim 2, in whichthe DNA sequence is obtainable from a DNA library of Aspergillusaculeatus, CBS 101.43.
 6. A DNA construct comprising the DNA sequence ofclaim
 1. 7. A recombinant expression vector comprising the DNA constructof claim
 6. 8. A host cell comprising a DNA construct according to claim6.
 9. A host cell comprising a recombinant expression vector of claim 7.10. The host cell of claim 8, which is a eukaryotic cell.
 11. The hostcell of claim 8, which a fungal cell.
 12. The host cell of claim 11,which is a yeast cell or a filamentous fungal cell.
 13. The host cell ofclaim 11, wherein the cell belongs to a strain of Aspergillus.
 14. Thehost cell of claim 11, wherein the cell belongs to a strain ofAspergillus niger or Aspergillus oryzae.
 15. A method of producing anenzyme exhibiting proteolytic activity comprising culturing the cell ofclaim 8 under conditions permitting the production of the enzyme, andrecovering the enzyme from the culture.
 16. An isolated and purifiedenzyme having the same activity as an enzyme having a pH optimum between4 and 7, exhibits protease activity in 5% hydrogen peroxide, isobtainable from filamentous fungus, and is encoded by a DNA sequencedepicted in SEQ ID NO: 1 or
 2. 17. The enzyme according to claim 16, inwhich enzyme is obtainable from a strain of Aspergillus, Rhizopus,Trichoderma, Penicillium, Fusarium, Schytalidium or Humicola.
 18. Theenzyme of claim 16, which is obtainable from a strain of Aspergillusaculeatus.